Daan Pieren

49 Novel features of ageing in murine T cells differences within naive or memory T cell populations. We indeed found inflation of the frequency of memory cells by aging. However, analysis of naive and the memory T cell pools revealed that attributing ageing-related differences to different T cell subsets should go beyond mere abundance of naive versus memory cells. Our phenotypic analyses show that the heterogeneity of both the naive and memory pools change with age. Among this heterogeneity, our ex vivo multi-dimensional analyses indicate that the naive CD4 + and Tc cell pools of aged mice are enriched for cells with a regulatory phenotype. CD122 + PD-1 + Tc cells have been reported to be Tc reg cells [41,42]. The high proportion of naive CD122 + PD-1 + Tc cells observed in aged mice indicates an accumulation of this regulatory cell type with ageing, which has, to the best of our knowledge, not been reported earlier. Additionally, the naive CD4 + T cell pool of aged mice was enriched for Foxp3 + Treg cells. In addition, aged Treg cells have previously been shown to contain higher proportions of PD-1 + cells. We now show that these PD-1 + Treg cells are present in both naive and memory Treg cell subsets. Moreover, higher proportions of CD25 low Treg cells at old age have been reported [40], and our data show that these cells are primarily present in the naive Treg cell subset. Our data analysis of combined expression of PD-1 and CD25 indicates a Treg subpopulation of PD-1 + CD25 Low CD4 + Tregs that increases with ageing. T cells at old age showed reduced IL-2 production and lack of proliferation. Since IL-2 is known for its capacity to promote T-cell proliferation, this finding suggested that the lack of IL-2 production may contribute to the lack of proliferation we observed at old age. However, addition of exogenous IL-2 did not result in restoration of T-cell proliferation in aged mice, indicating that reduced production of IL-2 is not likely to contribute to the reduced proliferation found at old age. Hence, explanations for the defective responsiveness should be searched for in other factors, such as the distorted expression of IL-2 receptor chains CD25 and CD122 [9,49,50] or increased co-inhibitory signals and increased Treg populations we observed at old age. Furthermore, aging-related mediators produced by non-T cells that may function as antigen presenting cells present in our cultures may have contributed to the aging-related defects of T-cell proliferation and activation we observed. Although altered co-stimulation by antigen-presenting cells reported with age may interfere with our findings [2,51], polyclonal stimulation of T cells may largely overrule aging-related effects on APC-mediated co-stimulation and antigen presentation in our assays. The strength of our analyses lies at examining the T-cell pool as a whole within its natural cell composition, which reflects the overall response of all splenocyte subpopulations. Many studies separate CD4 + and CD8 + naive and 2